Host defence peptides (HDPs) are innate immune effector molecules found in diverse species. HDPs exhibit a wide range of functions ranging from direct antimicrobial properties to immunomodulatory effects. Research in the last decade has demonstrated that HDPs are critical effectors of both innate and adaptive immunity. Various studies have hypothesized that the antimicrobial property of certain HDPs may be largely due to their immunomodulatory functions. Mechanistic studies revealed that the role of HDPs in immunity is very complex and involves various receptors, signalling pathways and transcription factors. This review will focus on the multiple functions of HDPs in immunity and inflammation, with special reference to cathelicidins, e.g. LL-37, certain defensins and novel synthetic innate defence regulator peptides. We also discuss emerging concepts of specific HDPs in immune-mediated inflammatory diseases, including the potential use of cationic peptides as therapeutics for immune-mediated inflammatory disorders.

The human host defense peptide LL-37 promotes immune activation such as induction of chemokine production and recruitment of leukocytes. Conversely, LL-37 also mediates anti-inflammatory responses such as production of anti-inflammatory cytokines, e.g., IL-1RA, and the control of pro-inflammatory cytokines, e.g., TNF. The mechanisms regulating these disparate immunomodulatory functions of LL-37 are not completely understood. Rho GTPases are GTP-binding proteins that promote fundamental immune functions such as chemokine production and recruitment of leukocytes. However, recent studies have shown that distinct Rho proteins can both negatively and positively regulate inflammation. Therefore, we interrogated the role of Rho GTPases in LL-37-mediated immunomodulation. We demonstrate that LL-37-induced production of chemokines, e.g., GRO-α and IL-8 is largely dependent on Cdc42/Rac1 Rho GTPase, but independent of the Ras pathway. In contrast, LL-37-induced production of the anti-inflammatory cytokine IL-1RA is not dependent on either Cdc42/Rac1 RhoGTPase or Ras GTPase. Functional studies confirmed that LL-37-induced recruitment of leukocytes (monocytes and neutrophils) is also dependent on Cdc42/Rac1 RhoGTPase activity. We demonstrate that Cdc42/Rac1-dependent bioactivity of LL-37 involves G-protein-coupled receptors (GPCR) and JNK mitogen-activated protein kinase (MAPK) signaling, but not p38 or ERK MAPK signaling. We further show that LL-37 specifically enhances the activity of Cdc42 Rho GTPase, and that the knockdown of Cdc42 suppresses LL-37-induced production of chemokines without altering the peptide’s ability to induce IL-1RA. This is the first study to demonstrate the role of Rho GTPases in LL-37-mediated responses. We demonstrate that LL-37 facilitates chemokine production and leukocyte recruitment engaging Cdc42/Rac1 Rho GTPase via GPCR and the JNK MAPK pathway. In contrast, LL-37-mediated anti-inflammatory cytokine IL-1RA production is independent of either Rho or Ras GTPase. The results of this study suggest that Cdc42 Rho GTPase may be the molecular switch that controls the opposing functions of LL-37 in the process of inflammation.

Introduction: Innate defence regulator (IDR) peptides are synthetic cationic peptides, variants of naturally occurring innate immune effector molecules known as host defence peptides. IDR peptides were recently demonstrated to limit infection-associated inflammation selectively without compromising host innate immune functions. This study examined the impact of a 12-amino acid IDR peptide, IDR-1002, in pro-inflammatory cytokine interleukin (IL)-1β-induced responses in synovial fibroblasts, a critical cell type in the pathogenesis of inflammatory arthritis. Methods: Human fibroblast-like synoviocytes (FLS) were stimulated with IL-1β in the presence and absence of IDR-1002. Production of enzyme matrix metalloproteinase-3 (MMP-3) and IL-1-receptor antagonist (IL-1RA) was monitored by enzyme-linked immunosorbent assay (ELISA), and various chemokines were evaluated by using multiplex cytometric bead array. Transcriptional responses were analyzed by quantitative real-time PCR. The impact on IL-1β-induced proteome was investigated by quantitative proteomics by using isobaric tags. IL-1β-induced pathways altered by IDR-1002 implicated by the proteomics analyses were further investigated by using various immunochemical assays. Cellular uptake of the peptide was monitored by using a biotinylated IDR-1002 peptide followed by microscopy probing with streptavidin-Alexa Fluor. Results: This study demonstrated that IDR-1002 suppressed the production of IL-1β-induced MMP-3 and monocyte chemotactic protein-1 (MCP-1); in contrast, IDR-1002 enhanced the production of IL-1RA, without neutralizing all chemokine responses. IDR-1002 altered the IL-1β-induced proteome primarily by altering the expression of members of nuclear factor kappa-B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The proteomics data also suggested that IDR-1002 was altering the transcription factor HNF-4α-mediated responses, known to be critical in metabolic regulation. With various immunochemical assays, it was further demonstrated that IL-1β-induced NF-κB, JNK, and p38 mitogen-activated protein kinase (MAPK) activations were significantly suppressed by IDR-1002. Conclusions: This study demonstrates the ability of an innate immune-modulatory IDR-peptide to influence the IL-1β-induced regulatory pathways and selectively to suppress inflammatory responses in synovial fibroblasts. The results of this study provide a rationale for examining the use of IDR-peptides as potential therapeutic candidates for chronic inflammatory diseases such as inflammatory arthritis.

Cytokines IL-32 and IL-17 are emerging as critical players in the pathophysiology of immune-mediated chronic inflammatory diseases. It has been speculated that the molecular mechanisms governing IL-32– and IL-17–mediated cellular responses are differentially dependent on the TNF pathway. In this study, kinome analysis demonstrated that following stimulation with cytokine IL-32, but not IL-17, there was increased phosphorylation of a peptide target corresponding to TNF-R1. Consistent with this observation, blocking TNF-R1 resulted in a suppression of IL-32–induced downstream responses, indicating that IL-32–mediated activity may be dependent on TNF-R1. In contrast, blocking TNF-R1 did not affect IL-17–induced downstream responses. Kinome analysis also implicated p300 (transcriptional coactivator) and death-associated protein kinase-1 (DAPK-1) as signaling intermediates for both IL-32 and IL-17. Phosphorylation of p300 and DAPK-1 upon stimulation with either IL-32 or IL-17 was confirmed by immunoblots. The presence of common targets was supported by results demonstrating similar downstream responses induced in the presence of IL-32 and IL-17, such as transcriptional responses and the direct activation of NF-κB. Furthermore, knockdown of p300 and DAPK-1 altered downstream responses induced by IL-32 and IL-17, and impacted certain cellular responses induced by TNF-α and IL-1β. We hypothesize that p300 and DAPK-1 represent nodes where the inflammatory networks of IL-32 and IL-17 overlap, and that these proteins would affect both TNF-R1–dependent and –independent pathways. Therefore, p300 and DAPK-1 are viable potential therapeutic targets for chronic inflammatory diseases.